The present invention relates to a method and a device for selecting accelerated proliferation of living cells in suspension.
Conventionally, it is distinguished between serial and continuous culture methods. In a serial culture technique, culture vessels containing a sterile growth medium are inoculated with a fraction of a culture which was grown under the same growth conditions. This cycle is repeated when the new culture has grown (Lenski, R. E. and Travisano, M. (1994): Dynamics of adaption and diversification: A 10,000-generation experiment with bacterial populations, Proc. Natl. Acad. Sci. USA 91, 6808-6814). The experiments described in the prior art in connection with the proliferation of microbial organisms over the longest time periods were carried out in accordance with this method (Lenski and Travisano, loc. cit.). In continuous culture methods (Dijkuizen, D. E. (1993): Chemostats used for studying natural selection and adaptive evolution, Meth. Enzymol. 224, 613-631) a culture is continuously diluted with a fresh growth medium in accordance with a predetermined regime. In the prior art such experiments are only described to last for limited time periods (Dijkuizen, loc. cit.).
The above-described conventional techniques are in particular disadvantageous in that so far no continuous culture method has been described which ensures the permanent proliferation of organisms in suspension. All described apparatuses select dilution-resistant (static) variants which populate the inner surfaces of the apparatus (Chao, L. and Ramsdell, G. (1985): The effects of wall populations on coexistence of bacteria in the liquid phase of chemostat cultures, J. Gen. Microbiol. 131, 1229-1236). These variants form a subpopulation which escapes the adaptive forces acting on the organisms in suspension. Continuous cultures with constant cell density, such as, e.g., turbidostat are particularly susceptible to an invasion by dilution-resistant variants and can only be carried out over relatively short time periods (about 200 generations). The prior art describes these difficulties but has so far only offered unsuitable solutions thereto. Therefore, such methods are not used in practice for scientific and industrial aims although their potential has been noticed very early (Monod, J. (1950): La technique de la culture continue. Thxc3xa9orie et applications, Ann. Inst. Pasteur 79, 390-410; Novick, A. and Szilard, L. (1950): Description of the chemostat, Science 112, 715-716). In the serial culture, which can be described as the periodical renewal of the culture apparatusxe2x80x94in the present case a simple culture vesselxe2x80x94the invasion with such dilution-resistant variants is avoided. However, it involves much work, i.e. many people are required and, due to the repeated manipulation under conditions which require absolute sterility, this culture is susceptible to contamination (Lenski and Travisano, loc. cit.). Robotization of the serial culture in a sterile environment could reduce these risks. However, it would be bought at the price that a large number of culture vessels are needed, and it would be limited since the mechanical precision of the robot and the sterile environment have to be maintained.
It is therefore an object of the present invention to provide an improved method and an improved device for selecting accelerated proliferation of living cells in suspension. This object is achieved with the features of the claims.
In achieving this object, the invention starts out from the basic idea that the device keeps a suspension of cells in continuous proliferation. A dilution-resistant variant must not be allowed to accumulate in any part of the apparatus. Its function is assured by controlling streams of liquid (fluidics). Under the prerequisite that the regular delivery of liquids, such as, e.g., nutrient media and washing solutions and a continuous supply with sterile gases, such as, e.g., air is assured, the apparatus must operate autonomously over an unlimited time period. Different culture regimes, such as, e.g., chemostat or turbidostat can be applied. If necessary, particular components of cells can be separated or isolated due to the effect of suitable solutions. If necessary, a plurality of these apparatuses can be combined with each other such that the content or part of the content of one apparatus can be transferred into another apparatus.
The requirement that under continuous culture conditions a population of cells only proliferates in suspension is concretely fulfilled by a preferably periodical transfer of the organism suspension from a first culture vessel into a second culture vessel. After the transfer, the first culture vessel has to undergo a sterilizing treatment, if necessary the sterilizing agent is neutralized, and the first culture vessel is then again ready for receiving the culture being transferred back from the second culture vessel, which is subsequently sterilized and neutralized.
This course of actions makes sure (i) that the population of organisms in suspension is maintained at any time and (ii) that all dilution-resistant variants in any part of the apparatus are destroyed during any one of the cycles.
The method of an alternating sterilization of the culture vessels preferably selects directly and regularly against variant organisms which populate the surfaces in the apparatus and avoids the proliferation and adaption of a static, dilution-resistant population. Any device for a continuous culture of organisms can be regarded as an apparatus in which the natural selection prefers mutants which resist dilution. The only possibility which the described method offers the cultivated population for withstanding dilution is an increase in the proliferation rate in suspension. In contrast to a fermentation method, the present invention describes an automated genetics method which simultaneously selects against static variants and prefers dynamic variants which are always better adapted to the culture conditions.
Thus, it is a particular advantage of the present invention vis-à-vis the prior art that a regime with a constant cell density (turbidostat) can be maintained over unlimited time periods and that the growth rate of naturally occurring cells or genetically modified cells can be increased. An example for an industrial application is the enrichment of natural variants being capable of metabolizing a chemical product (such as, e.g., an intermediate product of a chemical synthesis or an environmental pollutant). A further application would be the improvement of an enzyme or a metabolic pathway: if the conversion of a substrate into a product is the limiting step in the metabolism of a cell, and if the cell can be provided with a surplus of this substrate, under the described conditions the continuous culture will lead to an increase in the growth rate which results from an increased turnover rate of the substrate, and this increased turnover rate results from the fixation of the successive mutations in the gene or the genes for the enzymes which are subject to selection for the required turnover of the substrate.
It is a further advantage of the present invention that a plurality of different growth media can be supplied to the organism suspension by the apparatus. This makes it possible to carry out a multiple or alternating adaptation and to diversify the metabolic capacity of the cultivated organisms. A predetermined cell density can be made dependent on variables other than the supply of fresh medium. The fact that this density is reached can condition the effect of chemical and physical agents whose toxicity is adjusted such that the population of the organisms is always at its tolerance limit or that increasingly resistant variants are selected.
Moreover, due to the supply of detergents or solvents, certain components of cells can be extracted. In particular, genetic material such as plasmids or viruses can be extracted automatically, wherein the host cells are destroyed. However, particular agents which make the cells competent for genetic transformations or infections with naturally or synthetically produced nucleic acids can be supplied. This genetic material can then be introduced into the population. In very general terms, two or more apparatuses can be connected with each other. Thus, different organisms can be automatically brought in contact with each other and very different genetic materials such as, e.g., conjugative episomes, phages, transposons, etc. can be automatically introduced.